Immunotherapy in many different forms is making an increasingly strong impact on the treatment success in lymphoid malignancies. Monoclonal antibodies are a mainstay of treatment. In addition some novel agents, such as the immunomodulatory drugs thalidomide and lenalidomide appear to act primarily through an immune effect. In collaboration with the NCI lymphoma team, we have studied the effect of the monoclonal anti-CD20 antibody rituximab on tumor cells in patients with chronic lymphocytic leukemia (CLL). First, we identified about 80 genes up-regulated in tumor cells in response to rituximab infusion, many of which are known to be regulated by interferon (IFN) and to have pro-apoptotic function. We also found that IFN gamma was consistently upregulated in the serum within the first 6 hours of treatment indicating activation of immune effector cells. A surprising observation was that all these changes completely subsided by 24 hours. We hypothesized that this might be due to the process of CD20 shaving, a rapid and pronounced decrease of CD20 cell surface expression modeled in-vitro and in mice as the result of a mechanism called trogocytosis that relies on the direct and rapid exchange of cell membrane fragments and associated molecules between effectors and target cells (Beum, J Immunol, 2008). First, we used Western blot analysis of total CD20 protein in CLL cells and found a rapid loss of CD20 that was apparent already at 2 hours from the start of rituximab resulting in virtually complete loss of expression at 24 hours. Next, we used ImageStream technology to directly visualize tumor-immune cell interactions in-vivo. We found transfer of CD20 from CLL cells to NK cells and monocytes, resulting in complete CD20 loss in circulating CLL cells. While we detected transfer of CD20 into both cell types, monocytes were much more engaged in trogocytosis than NK cells. CD20 shaving appears to be a mechanism how tumor cells can resist treatment given that that persistent disease in the bone marrow was mostly CD20 negative. In parallel we also investigated changes in the function of NK cells and monocytes: consistent with NK cell activation we found an increase in CD69 after RTX administration and down-regulation of perforin expression. Activation of NK cells is triggered by the engagement of CD16/FcRIIIa by rituximab coated CLL cells. Interestingly, CD16 expression on NK cells was rapidly lost and was not completely recovered by 24 hours. In addition to loss of CD16, we found that the cytotoxic capacity of the effector cells was rapidly exhausted: both NK cells and monocytes isolated from patients during treatment showed a significant decrease in their ability to kill tumor cells. Collectively, our results identify loss of CD20 from CLL cells and exhaustion of immune effector mechanisms as limitations for anti-CD20 immunotherapy. We have recently initiated a new treatment trial for CLL patients using ofatumumab, a 2nd generation anti CD20 antibody. Preliminary results demonstrate that ofatumumab is stronger in activating complement mediated killing of CLL cells than rituximab, but it appears to also cause loss of CD20 on tumor cells. These data identify possible avenues for improving CD20 mediated immunotherapy and characterize endpoints on which different anti-CD20 antibodies can be compared. These mechanisms may be of general importance to immunotherapy of hematologic malignancies. Following up on gene expression data that had identified a gene called TOSO as highly overexpressed in CLL cells, we generated protein-drug conjugates that can selectively target CLL cells. TOSO is a transmembrane protein that is expressed on some normal B-cells but is highly overexpressed on CLL cells. We discovered that TOSO can rapidly uptake IgM, internalize it in specific vesicles, and transport it through the endocytic pathway to the lysosomes. Interestingly, aggregation of TOSO with IgM lead to rapid internalization of IgM whereas mAb bound TOSO was not internalized. Because of its restricted expression on CLL cells and its intracellular trafficking, we think that TOSO represents a potential means of delivering a cytotoxic agent into malignant cells. To this end, we engineered a protein backbone derived from IgM that can be conjugated to cytotoxic drugs. We have now shown that such constructs can selectively kill TOSO expressing cells in vitro and in vivo. Having shown proof of concept for this approach, we are now working on optimizing drug to carrier ratios and evaluate a set of different drugs to be conjugated to the protein carrier. These preclinical studies set the stage for possible clinical exploration of this approach as a treatment for CLL. Anti-CD20 monoclonal antibodies (mAbs) are an important component of treatment regimens for B-cell malignancies. Following administration of anti-CD20 mAbs CD20 expression is down-modulated by trogocytosis and internalization. While cells with low or absent CD20 expression can evade further anti-CD20 mAb binding, they are labelled by complement proteins. In particular, complement component C3d is covalently bound to the cell surface. We hypothesized that C3d constitutes a neoantigen that could be targeted by anti-C3d mAbs to enhance anti-CD20 therapy. We derived several hybridomas from mice immunized with human C3d and found to bind three distinct epitopes on C3d. One epitope was shared by all high affinity mAbs, while two other epitopes were bound by low affinity mAbs. From a high affinity murine mAb we derived a human IgG1 chimeric antibody that is highly selective for C3d, does not bind full length C3, and does not cross react with mouse C3d. The chimeric anti-C3d mAb bound immobilized C3d with a Kd of 17nM and cell-bound C3d with a kD of 3.0nM, similar to the kD of 2.4nM of ofatumumab binding to CD20. The anti-C3d antibody mediated complement-dependent cytotoxicity, NK cellular cytotoxicity, and phagocytosis of C3d opsonized cells. Repeat targeting of C3d opsonized cells in the presence of human serum in vitro resulted in increased deposition of C3d leading to a 5-fold increase of binding sites per cell resulting in an auto-amplification loop and the killing of >95% of cells through complement-dependent lysis. We tested activity of the anti-C3d mAb against primary tumor cells from patients with chronic lymphocytic leukemia (CLL) being treated with the anti-CD20 mAb ofatumumab. The first administration of ofatumumab decreased the tumor cell count in the blood of patients by 50% on average. As expected, CLL cells obtained 24 hours after administration of ofatumumab had lost CD20 antigen and carried abundant C3d on their cell surface. Importantly, the anti-C3d chimeric antibody specifically bound CLL cells but no other blood cells, indicating that targeting of C3d preserves the specificity of the initial complement fixing antibody. These studies provide proof of concept that targeting cell deposited C3d can enhance the potency of mAb therapy. Given that our anti-C3d mAb preserves the specificity of the initial mAb, it could augment the potency of many mAbs currently in clinical use by delivering a one-two punch. We conclude that targeting C3d deposited on cancer cells can eliminate antigen escape variants and potentiate existing therapeutic antibodies.